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NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Therapies targeting TIGIT have garnered tremendous interest, but have so far failed to reach primary endpoints in PhIII trials in lung cancer settings. This study examined the function of TIGIT on NK cells and how NK cells may impact the success of TIGIT therapeutics. This study demonstrated that TIGIT expression is increased on activated NK cells, including those expanded with clinical protocols for cell therapy. Activated TIGIT+ NK cells have a better anti-tumor response as compared to TIGIT− NK cells. More importantly, higher tumor infiltration of activated NK cells correlates with better patient outcomes in lung adenocarcinoma. However, chronic TIGIT engagement with its ligands in the tumor microenvironment leads to the functional decline of NK cells, which can be prevented with anti-TIGIT. This demonstrates that TIGIT-expressing cells are not inherently exhausted but can represent NK cells with the highest anti-tumor activity. These findings support the joint application of NK cells with blocking, non-depleting anti-TIGIT for improved treatment outcomes. Treatments targeting TIGIT have gained a lot of attention due to strong preclinical and early clinical results, particularly with anti-PD-(L)1 therapeutics. However, this combination has failed to meet progression-free survival endpoints in phase III trials. Most of our understanding of TIGIT comes from studies of T cell function. Yet, this inhibitory receptor is often upregulated to the same, or higher, extent on NK cells in cancers. Studies in murine models have demonstrated that TIGIT inhibits NK cells and promotes exhaustion, with its effects on tumor control also being dependent on NK cells. However, there are limited studies assessing the role of TIGIT on the function of human NK cells (hNK), particularly in lung cancer. Most studies used NK cell lines or tested TIGIT blockade to reactivate exhausted cells obtained from cancer patients. For therapeutic advancement, a better understanding of TIGIT in the context of activated hNK cells is crucial, which is different than exhausted NK cells, and critical in the context of adoptive NK cell therapeutics that may be combined with TIGIT blockade. In this study, the effect of TIGIT blockade on the anti-tumor activities of human ex vivo-expanded NK cells was evaluated in vitro in the context of lung cancer. TIGIT expression was higher on activated and/or expanded NK cells compared to resting NK cells. More TIGIT+ NK cells expressed major activating receptors and exerted anti-tumor response as compared to TIGIT− cells, indicating that NK cells with greater anti-tumor function express more TIGIT. However, long-term TIGIT engagement upon exposure to PVR+ tumors downregulated the cytotoxic function of expanded NK cells while the inclusion of TIGIT blockade increased cytotoxicity, restored the effector functions against PVR-positive targets, and upregulated immune inflammation-related gene sets. These combined results indicate that TIGIT blockade can preserve the activation state of NK cells during exposure to PVR+ tumors. These results support the notion that a functional NK cell compartment is critical for anti-tumor response and anti-TIGIT/adoptive NK cell combinations have the potential to improve outcomes. [ABSTRACT FROM AUTHOR]

There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity in vitro using 2D and 3D cancer models and in vivo in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ. In vivo antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh vs. cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both in vitro and in vivo. These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy is a highly successful and rapidly evolving treatment modality that works by augmenting the body's own immune system. While various immune stimulation strategies such as PD-1/PD-L1 or CTLA-4 checkpoint blockade result in robust responses, even in patients with advanced cancers, the overall response rate is low. While immune checkpoint inhibitors are known to enhance cytotoxic T cells' antitumor response, current evidence suggests that immune responses independent of cytotoxic T cells, such as Natural Killer (NK) cells, play crucial role in the efficacy of immunotherapeutic interventions. NK cells hold a distinct role in potentiating the innate immune response and activating the adaptive immune system. This review highlights the importance of the early actions of the NK cell response and the pivotal role NK cells hold in priming the immune system and setting the stage for successful response to cancer immunotherapy. Yet, in many patients the NK cell compartment is compromised thus lowering the chances of successful outcomes of many immunotherapies. An overview of mechanisms that can drive NK cell dysfunction and hinder immunotherapy success is provided. Rather than relying on the likely dysfunctional endogenous NK cells to work with immunotherapies, adoptive allogeneic NK cell therapies provide a viable solution to increase response to immunotherapies. This review highlights the advances made in development of NK cell therapeutics for clinical application with evidence supporting their combinatorial application with other immune-oncology approaches to improve outcomes of immunotherapies. [ABSTRACT FROM AUTHOR]

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline- II motifs are often located near sites of phosphorylation. Wehave used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus nonstructural protein 5A (NS5A) on Thr-2332 near one of its polyproline- II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using 15N-, 13C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitisCvirus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome. [ABSTRACT FROM AUTHOR]

Background: Inhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT + immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied., Methods: CRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT., Results: TIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT., Conclusions: TIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy., Competing Interests: Competing interests: AJC: licensed IP to, consultancy and research support from Kiadis Pharma, a Sanofi company; JLO: licensed IP to, consultancy with Kiadis Pharma, a Sanofi company. MFH, TJC-P: licensed IP to Kiadis Pharma, a Sanofi company; DL: scientific advisory board of and consults for Avidicure; consultancy, licensing, and royalty fees from Kiadis Pharma, a Sanofi Company; IP interests related to NK-cell therapy., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)